Lentiviral LTR-deleted vector

ABSTRACT

A vector capable of transducing non-dividing and/or slowly dividing cells is provided, wherein the vector is a lentiviral LTR-deleted vector. Also provided is a method for producing a protein of interest in a non-dividing or slowly dividing cell by transducing the cell with a lentiviral LTR-deleted vector and expressing the protein of interest in the cell. In addition, target cells containing the lentiviral LTR-deleted vector are provided.

RELATED APPLICATIONS

[0001] This application is a continuation-in-part of U.S. applicationSer. No. 09/254,832, filed on Jun. 21, 1999, as the national phaseapplication of International application Serial No. PCT/GB97/02969,filed on Oct. 28, 1997 and claiming priority to UK application SerialNo. GB 9622500.8, filed on Oct. 29, 1996. This application makesreference to U.S. Pat. No. 6,235,522, filed on Apr. 5, 1999 as thenational phase application of International application Serial No.PCT/GB97/02858, filed on Oct. 17, 1997 and claiming priority to UKapplication Serial No. GB 9621680. This application also makes referenceto: U.S. Pat. No. 6,096,538, filed on Nov. 19, 1997, U.S. Pat. No.6,132,731, filed on Oct. 8,1997, U.S. Pat. No. 6,168,916, filed on Oct.21, 1998, U.S. Pat. No. 6,312,682, filed on Dec. 28, 1998, U.S. Pat. No.6,312,683, filed on Jan. 27, 1999, U.S. application Ser. No. 09/533,276,filed on Mar. 22, 2000, U.S. application Ser. No. 09/533,295, filed onMar. 22, 2000, U.S. application Ser. No. 09/552,950, filed on Apr. 20,2000, U.S. application Ser. No. 09/860,996, filed on May 18, 2001, U.S.application Ser. No. 09/867,947, filed on May 29, 2001, U.S. applicationSer. No. 09/915,169, filed on Jul. 25, 2001, U.S. application Ser. No.10/001,220, filed on Nov. 15, 2001, U.S. application Ser. No.10/002,598, filed on Nov. 15, 2001, and U.S. application Ser. No.10/008,610, filed on Nov. 8, 2001.

[0002] Each document cited or referenced in each of the foregoingapplications, and any manufacturer's instructions or catalogues for anyproducts cited or mentioned in each of the foregoing applications and inany of the cited documents, are hereby incorporated herein by reference.Furthermore, all documents cited in this text, all documents cited orreferenced in documents cited in this text, and any manufacturer'sinstructions or catalogues for any products cited or mentioned in thistext or in any document incorporated into this text, are incorporatedherein by reference. Documents incorporated by reference into this textor any teachings therein can be used in the practice of this invention.Documents incorporated by reference into this text are not admitted tobe prior art.

FIELD OF THE INVENTION

[0003] This invention relates to lentiviral long terminal repeat(LTR)-deleted vectors. The invention also relates to lentiviralLTR-deleted vectors carrying nucleotide sequences of interest, and totheir use in transferring genetic material to non-dividing or slowlydividing cells.

BACKGROUND OF THE INVENTION

[0004] Amongst nucleic acid transfer systems, retroviral vectors holdsubstantial promise for gene therapy and other applications in whichtransfer of genetic material is desirable. These systems can transfergenes efficiently, and new vectors are emerging that are particularlyuseful for gene delivery to brain cells (Naldini et al., 1996 Science272, 263).

[0005] There has been considerable interest in the development ofretroviral vector systems based on lentiviruses, a small subgroup of theretroviruses. This interest arises firstly from the notion of usingHIV-based vectors to target anti-HIV therapeutic genes to HIVsusceptible cells and secondly from the prediction that, becauselentiviruses are able to infect non-dividing cells (Lewis & Emerman 1993J.Virol. 68, 510), vector systems based on these viruses are able totransduce non-dividing cells (e.g. Vile & Russel 1995 Brit. Med. Bull.51, 12). Vector systems based on HIV have been produced (Buchschacher &Panganiban 1992 J.Virol. 66, 2731) and have been used to transduce CD4+cells and non-diving cells (Naldini et al., 1996 Science 272, 263).However, in general, nucleic acid transfer efficiencies are not as highas with comparable murine retrovirus vector systems.

[0006] The HIV-based vectors produced to date result in an integratedprovirus in the transduced cell that has HIV LTRs at its ends. Thislimits the use of these vectors as the LTRs have to be used asexpression signals for any inserted gene unless an internal promoter isused. The use of internal promoters has significant disadvantages. Forexample, the presence of internal promoters can affect the transductiontitres obtainable from a packaging cell line and the stability of theintegrated vector.

[0007] Also, HIV and other lentiviral LTRs have virus-specificrequirements for nucleic acid expression. For example, the HIV LTR isnot active in the absence of the viral Tat protein (Cullen 1995 AIDS 9,S19). It is desirable, therefore, to modify or delete the LTRs in such away as to change the requirements for nucleic acid expression. Inparticular, tissue specific gene expression signals may be required forsome gene therapy applications. In addition, signals that respond toexogenous signals may be necessary. In murine retroviruses this is oftenachieved simply by replacing the enhancer-like elements in the U3 regionof the murine lentiviral (MLV) LTR by enhancers that respond to thedesired signals. This has not been feasible with viruses such as HIVbecause within the U3 and R regions of their LTRs are sequences, knownas IST and TAR, which may inhibit gene expression and may or may not beresponsive to Tat protein when heterologous, perhaps tissue specific,control sequences are inserted in the U3 region (Cullen 1995 AIDS 9,S19; Alonso et al., 1994 J. Virol. 68, 6505; Ratnasabapathy et al., 19904, 2061;Sengupta et al., 1990 PNAS 87, 7492; Parkin et al., 1988 EMBO.J7, 2831). Even if the signals are responsive, it is undesirable to haveto supply Tat as it further complicates the system and Tat has someproperties of oncoproteins (Vogel et al., 1988 Nature 335, 606).

[0008] Parkinson's disease (PD) is a common neurodegenerative disorderthat afflicts the growing population of elderly people. Patients displaytremor, cogwheel rigidity and impairment of movement. It is generallythought to be an acquired rather than inherited disease in whichenvironmental toxins, metabolic disorders, infectious agents and normalaging have all been implicated. PD is associated with the degenerationof nigrostriatal neurons which have their soma located in the substantianigra. They send axonal projections to the basal ganglia and they usedopamine as their neurotransmitter. Some features of the disease can becontrolled by the administration of L-DOPA, the metabolic precursor todopamine, which diffuses across the blood brain barrier more effectivelythan dopamine itself. Unfortunately as the disease progresses the sideeffects of this treatment become unacceptable.

[0009] PD is an ideal candidate for gene therapy for several reasons.The clinical efficacy of systemic administration of L-DOPA suggests thatrestoration of neuronal circuitry is not essential for diseasemanagement. Therefore genetic manipulation of brain cells to providelocal production of L-DOPA from tyrosine may be a realistic strategy fortreatment. The biosynthesis of L-DOPA from tyrosine involves a singlestep suggesting that provision of tyrosine hydroxylase (TH) by geneticmeans may be sufficient and some success has been achieved using thisstrategy in small animals and in cell culture (Kaplitt et al., 1994Nature Genetics 8, 148; During et al., 1994 Science 266, 1399; Horellouet al., 1994 Neuroreport 6, 49; Owens et al., 1991 J. Neurochem. 56,1030). However, if one is to use local endogenous brain cells as L-DOPAfactories for the treatment of PD in man it is likely that high levelsof L-DOPA will be required to effect a treatment. These high levels mustbe efficiently converted to dopamine as the necessary neurotransmitterand primary therapeutic agent. It is likely therefore that it will benecessary not only to supply tyrosine hydroxylase but also DOPAdecarboxylase (DD), the enzyme that converts L-DOPA to dopamine. Thismeans that in a gene therapy strategy the genes for both of theseenzymes will be required. However, it is clear from the literature thatretroviral vectors achieve the highest titres and most potent geneexpression properties if they are kept genetically simple(PCT/GB96/01230; Bowtell et al., 1988 J.Virol. 62, 2464; Correll et al.,1994 Blood 84, 1812; Emerman and Temin 1984 Cell 39, 459; Ghattas etal., 1991 MoI.CeII.Biol. 11, 5848; Hantzopoulos et al., 1989 PNAS 86,3519; Hatzoglou et al., 1991 J.BioI.Chem 266, 8416; Hatzoglou et al.,1988 J.BioI.Chem 263, 17798; Li et al., 1992 Hum.Gen.Ther. 3, 381;McLachlin et al., 1993 Virol.195, 1; Overell et al., 1988 MoI.Cell Biol.8, 1803; Scharfman et al., 1991 PNAS 88, 4626; Vile et al., 1994 GeneTher 1, 307; Xu et al., 1989 Virol. 171, 331; Yee et al., 1987 PNAS 84,5197). This means only using a single transcription unit within thevector genome and orchestrating appropriate nucleic acid expression fromsequences within the 5′ LTR. The need to express two enzymes from asingle retroviral vector would require the use of an internal ribosomeentry site (IRES) to initiate translation of the second coding sequencein a poly-cistronic message (Adam et al 1991 J.Virol. 65, 4985).However, the efficiency of an IRES is often low and tissue dependentmaking this strategy undesirable when one is seeking to maximise theefficiency of metabolic conversion of tyrosine through to dopamine. Thepresent invention addresses these problems.

SUMMARY OF THE INVENTION

[0010] The present invention provides in one aspect a lentiviral vectorcapable of transducing a non-dividing or slowly-dividing cell, saidvector comprising a lentiviral LTR-deleted vector. The vector canfurther comprise a nucleotide sequence encoding a polypeptide or proteinof interest (POI), e.g., at least one nucleotide sequence of interest(NOI) encoding at least one POI. Advantageously, the NOI is operablylinked to a promoter. If there is more than one NOI, there can be onepromoter for driving expression, or a promoter for each NOI for drivingexpression. Thus, one or more NOI can be operably linked to one or moreNOI. The vector can comprise a polynucleotide sequence, which encodestwo or more POI, e.g., therapeutic POI, operably linked to a promoter,and the polynucleotide can encode a fusion POI. The invention thus canprovide a way of expressing two therapeutic NOI from a single “chimeric”gene or polynucleotide. The vector may be for example an expressionvector such as a plasmid, or it may be a retroviral vector particlecomprising an RNA genome containing the nucleotide sequences asdescribed herein.

[0011] In another aspect, the invention provides a method for producinga POI in a non-dividing or slowly-dividing cell, comprising transducingthe cell with a lentiviral LTR-deleted vector and expressing the POI inthe cell. In a preferred embodiment, the non-dividing cell is a neuron.

[0012] There are many uses for in vitro expressed POI. For instance,depending on the nature of the POI, the in vitro expressed POI canrepresent a protein that is purer than if the POI was isolated from itsnative environment, as it would be free from contaminants from thatenvironment. Thus, such POI can be used in assays, to generateantibodies, e.g., for use in assays, as antigens or epitopes inimmunological compositions, and as active agents in therapeutic,pharmaceutical or veterinary compositions, inter alia.

[0013] The invention further provides a target cell in vitro comprisinga lentiviral LTR-deleted vector. In yet further aspects, the inventionprovides a DNA construct encoding the RNA genome for the retroviralvector particle; and a retroviral vector production system comprising aset of nucleic acid sequences encoding the components of the retroviralvector particle.

[0014] The invention further provides the use of retroviral vectorscarrying the chimeric gene described herein, in gene therapy and in thepreparation of a medicament for gene therapy; and a method of performinggene therapy on a target cell, which method comprises transducing thetarget cell with a lentiviral LTR-deleted vector comprising a nucleotidesequence encoding a POI, thus delivering the nucleotide sequence to thetarget cell. The invention further provides transduced target cellsresulting from these methods and uses. The invention thus provides agene delivery system for use in medicine.

[0015] The term “comprising” in this disclosure can mean “including” orcan have the meaning commonly given to the term “comprising” in U.S.Patent Law.

[0016] Other aspects of the invention are described in or are obviousfrom (and within the ambit of the invention) the following disclosure.

BRIEF DESCRIPTION OF DRAWINGS

[0017] The following Detailed Description, given by way of example, butnot intended to limit the invention to specific embodiments described,may be understood in conjunction with the accompanying drawings,incorporated herein by reference, in which:

[0018]FIG. 1 shows a general scheme for Lentiviral LTR-deleted (LLD)vectors which may be used with the present invention and which areemployed in the Examples.

[0019]FIG. 2 shows a generalised HIV-based LLD vector genome asdescribed in the Examples; Superscript H=HIV-derived sequence (could befrom any lentivirus); Superscript M=MLV-derived sequence; 1V=Packagingsite (including gag region); PBS=Second strand priming site;INTERNAL=Region containing genes, selectable markers, other promoters orRNA handling systems such as HIV RRE and Rev coding sequences.

[0020]FIG. 3 shows a specific HIV-based LLD vector genome as describedin the Examples. NIT vector genome (Inserts 3789bp+backbone 2929bp=6718bp): HCMV promoter (−521 to −1) from pRV109; HIV sequences (552 to 1144;5861 to 6403; 7621 to 9085) from HXB2; geonotype:gag−;pol−;env−;rev+;RRE; vif−;vpu−;vpr−;tat−;nef−; mutations; threepoint mutations to remove ATG (790, 834, 894) (@) a frameshift mutationby two base insertion (831) (*); a deletion between NdeI (6403) andBgIII (7621) (Δ); polycloning site (X);XhoI-SalI-ClaI-EcoRIV-EcoRI-PstI-SmaI-SmaI-BamHI-SpeI (underlined sitesare unique); maximal insertion size into the polycloning site: 5997bp;backbone: pBluescriptKS+

[0021]FIG. 4 shows in detail the structure of the 3′ LTR for the vectorin FIG. 3.

[0022]FIG. 5 shows a schematic diagram of packaging components suitablefor use with the vector genome shown in FIGS. 1 to 3. pRV664 encodesHIV-1 HXB2 gagpol (637-5748) and contains RRE (77208054) and itsbackbone is pCI-neo (PROMEGA). pRV438 possesses both rev and env fromHXB2 (5955-8902) in pSA91 which is a mammalian expression plasmid withCMC promoter. pSyngp 160 mn (from B. Seed) is an expression plasmid forHIV-1 MN envelope which was modified to have the optimized codon usagein mammalian cells. pRV67 is a VSV G expression plasmid in pSA91.

[0023]FIG. 6 further shows the principle of vectors according to thisinvention.

[0024]FIG. 7 shows simplified directions for construction ofpolynucleotide sequences according to the invention, encoding TH-DDfusion proteins.

[0025]FIG. 8 shows simplified directions for construction ofpolynucleotide sequences according to the invention, encoding DD-THfusion proteins.

[0026]FIG. 9 shows primers for use in the construction methodsillustrated in FIGS. 7 and 8 and described in detail in the Examples.Lower case nucleotides denote rare codons in highly expressed genes inmammalian cells (Haas et al., 1996 Cur. Biol. 6, 315).

DETAILED DESCRIPTION OF THE INVENTION

[0027] The lentivirus of the invention provides the ability to infectand transduce non-dividing and/or slowly-dividing cells. During theinfection process, lentiviruses form a pre-integration complex in thetarget cell cytoplasm containing integrase, core proteins and theproviral DNA. The complex is able to pass across the nuclear membrane ofthe target cell, by means of signal sequences in the proteins. Otherretroviruses either lack the proteins, or have the proteins but withoutthe appropriate signal sequences. It is therefore expected to bepossible in principle to introduce into retroviruses other thanlentiviruses the ability to infect non-dividing or slowly-dividingcells.

[0028] To date, the most widely used retroviral vector systems for humangene therapy applications have used MLV. However, retroviral vectorsystems may also be based on other oncoretroviruses (the sub-group ofretroviruses containing MLV), lentiviruses, or retroviruses from othersub-groups. Examples of lentiviruses are HIV, SIV, FIV, BLV, EIAV, CAEVand visna virus. Of these, HIV and SIV are presently best understood.However, preferred for use in gene therapy would be anon-immunodeficiency lentivirus because the immunodeficiency virusesinevitably bring with them safety considerations and prejudices. A rangeof retroviruses have already been split into packaging and vectorcomponents for retroviral vector particle production systems, includingASLV, SNV and RSV. It will be evident that a retroviral vector accordingto the invention need not be confined to the components of a particularretrovirus. The retroviral vector may comprise components derived fromtwo or more different retroviruses, and may also comprise syntheticcomponents. Vector components can be manipulated to obtain desiredcharacteristics, such as target cell specificity.

[0029] The lentivirus group can be split into “primate” and“non-primate”. Examples of primate lentiviruses include the humanimmunodeficiency virus (HIV), the causative agent of human acquiredimmunodeficiency syndrome (AIDS), and the simian immunodeficiency virus(SIV). The non-primate lentiviral group includes the prototype “slowvirus” visna/maedi virus (VMV), as well as the related caprinearthritis-encephalitis virus (CAEV), equine infectious anaemia virus(EIAV) and the more recently described feline immunodeficiency virus(FIV) and bovine immunodeficiency virus (BIV).

[0030] Details on the genomic structure of some lentiviruses may befound in the art. By way of example, details on HIV and EIAV may befound from the NCBI Genbank database (i.e. Genome Accession Nos.AF033819 and AF033820 respectively). Details of HIV variants may also befound at http://hiv-web.lanl.gov. Details of EIAV variants may be foundthrough http://www.ncbi.nlm.nih.gov. Further details on EIAV can befound in U.S. Pat. No. 6,277,633, incorporated herein by reference.

[0031] Lentiviruses that are the subject of patents and patentpublications and patent applications of Oxford Biomedica areadvantageously employed in the practice of the invention.

[0032] During the process of infection, a retrovirus initially attachesto a specific cell surface receptor. On entry into the susceptible hostcell, the retroviral RNA genome is then copied to DNA by the virallyencoded reverse transcriptase which is carried inside the parent virus.This DNA is transported to the host cell nucleus where it subsequentlyintegrates into the host genome. At this stage, it is typically referredto as the provirus. The provirus is stable in the host chromosome duringcell division and is transcribed like other cellular genes. The provirusencodes the proteins and other factors required to make more virus,which can leave the cell by a process sometimes called “budding”.

[0033] Each retroviral genome comprises genes called gag, pol and envwhich code for virion proteins and enzymes. These genes are flanked atboth ends by regions called long terminal repeats (LTRs). The LTRs areresponsible for proviral integration, and transcription. They also serveas enhancer-promoter sequences. In other words, the LTRs can control theexpression of the viral genes. Encapsidation of the retroviral RNAsoccurs by virtue of a psi sequence located at the 5′ end of the viralgenome.

[0034] The LTRs themselves are identical sequences that can be dividedinto three elements, which are called U3, R and U5. U3 is derived fromthe sequence unique to the 3′ end of the RNA. R is derived from asequence repeated at both ends of the RNA, and U5 is derived from thesequence unique to the 5′end of the RNA. The sizes of the three elementscan vary considerably among different retroviruses.

[0035] For the viral genome, the site of transcription initiation is atthe boundary between U3 and R in the left hand side LTR and the site ofpoly (A) addition (termination) is at the boundary between R and U5 inthe right hand side LTR. U3 contains most of the transcriptional controlelements of the provirus, which include the promoter and multipleenhancer sequences responsive to cellular and in some cases, viraltranscriptional activator proteins. Some retroviruses have any one ormore of the following genes that code for proteins that are involved inthe regulation of gene expression: tat, rev, tax and rex.

[0036] With regard to the structural genes gag, pol and env themselves,gag encodes the internal structural protein of the virus. Gag protein isproteolytically processed into the mature proteins MA (matrix), CA(capsid) and NC (nucleocapsid). The pol gene encodes the reversetranscriptase (RT), which contains DNA polymerase, associated RNase Hand integrase (IN), which mediate replication of the genome. The envgene encodes the surface (SU) glycoprotein and the transmembrane (TM)protein of the virion, which form a complex that interacts specificallywith cellular receptor proteins. This interaction leads ultimately toinfection by fusion of the viral membrane with the cell membrane.

[0037] Lentiviruses may also contain “additional” genes which code forproteins other than gag, pol and env. Examples of additional genesinclude in HIV, one or more of vif vpr, vpx, vpu, tat, rev and nef. EIAVhas, for example, the additional genes S2 and dUTPase.

[0038] Proteins encoded by additional genes serve various functions,some of which may be duplicative of a function provided by a cellularprotein. In EIAV, for example, tat acts as a transcriptional activatorof the viral LTR. It binds to a stable, stem-loop RNA secondarystructure referred to as TAR. Rev regulates and co-ordinates theexpression of viral genes through rev-response elements (RRE). Themechanisms of action of these two proteins are thought to be broadlysimilar to the analogous mechanisms in the primate viruses. The functionof S2 is unknown. In addition, an EIAV protein, Ttm, has been identifiedthat is encoded by the first exon of tat spliced to the env codingsequence at the start of the transmembrane protein.

[0039] For the production of retroviral vector particles, the vector RNAgenome is expressed from a DNA construct encoding it, in a host cell.The components of the particles not encoded by the vector genome areprovided in trans by additional nucleic acid sequences (the “packagingsystem”, which usually includes either or both of the gag/pol and envgenes) expressed in the host cell. The set of sequences required for theproduction of the retroviral vector particles may be introduced into thehost cell by transient transfection, or they may be integrated into thehost cell genome, or they may be provided in a mixture of ways. Thetechniques involved are known to those skilled in the art.

[0040] Certain retroviruses have special characteristics which may beuseful in particular gene therapy applications. For example, thelentiviruses such as HIV are capable of infecting and transducingnon-dividing and/or slowly-dividing cells because they have means forgetting the proviral DNA across the nuclear membrane of target cells.This feature will be useful if it is desired to target non-dividing orslowly-dividing cell types in nucleic acid transfer. Such cell typesinclude the neurons of the human brain, which are a potentiallyimportant target for gene therapy treatment of Parkinson's disease. Aretroviral vector particle according to the invention may thus bederived from a lentivirus, at least to the extent that it is capable ofdelivering proviral DNA efficiently to a non-dividing or slowly-dividingcell.

[0041] The vector can comprise a non-lentiviral expression controlelement, which will usually be a promoter. This term includes knownpromoters, in part or in their entirety, which may be constitutivelyacting or may be inducible only under certain conditions e.g. in thepresence of a regulatory protein. This enables expression of one or moreNOI to be restricted e.g. to particular cell types or to cells in whicha particular exogenous signal is present. For example, heavy metalinduction of a NOI could be achieved by using components of themetallothionein promoter. Expression control by a steroid hormone may beanother useful approach. Brain-specific, stem cell specific ortumour-specific gene expression signals might alternatively be used.

[0042] The non-lentiviral promoter replaces the lentiviralprotein-dependent promoter function of the lentiviral 5′ LTR. For HIV,this means that the 5′ LTR is no longer responsive to the HIV Tatprotein. Tat acts on the TAR region of R; in an HIV-based vectoraccording to the invention functional TAR sequences are therefore absentin order to avoid reductions of translation by the TAR structure.Enhancer sequences contained in the HIV U3 regions are also preferablyexcluded. A straightforward way to achieve the desired vector LTRs istherefore to replace the lentiviral R regions and as far as possible theU3 regions, but leaving essential lentiviral sequences present such as ashort sequence of the U3 region necessary for integration.

[0043] The invention is outlined in FIG. 1. The vector system isdesignated Lentiviral LTR-Deleted (LLD) vector. It comprises a DNAmolecule in which a CMV or other high efficiency promoter is used todrive the expression of the vector RNA in a producer cell. This strategyis analogous to the HIT vector system (Soneoka et al., 1995 Nucl.AcidsRes. 23, 628). The producer cell will have been engineered to producecompatible lentiviral structural proteins and enzymes. It will be,therefore, what is known as a vector packaging cell. The producer DNAcan be used as an autonomous plasmid that either does or does notreplicate or it can be integrated into the producer cell genome. All ofthese strategies are known in the field (Soneoka et al., 1995 Nucl.AcidsRes. 23, 628; Miller and Rossman 1989 BioTech. 7, 980; Miller 1990Hum.Gene Ther. 1, 5). The producer DNA for the vector genome may containat least the following contiguous components: a high efficiencypromoter; a non-lentiviral R region that either comes from anotherretrovirus or is completely synthetic; all or part of the lentiviral U5region that contains sequences required for integration by thelentiviral integrase and sequences necessary for efficient reversetranscription; packaging signals that are recognized by the packagingcomponents of the producer cell; an internal region that might containone or more NOI, including therapeutic or reporter NOI or selectablemarkers and associated expression signals (in addition the internalregion might contain components of systems for ensuring efficient RNAsplicing and transport); a second strand primer site from thelentivirus; a short sequence of 30-100 nucleotides from the lentivirusU3 region that is required for efficient integration by the lentivirusintegrase; a heterologous promoter that might confer tissue specificityof gene expression or regulation by an exogenous signal so that a NOIcan be expressed appropriately; and an R region that is identical to thefirst R region together with transcription termination andpolyadenylation signals required to produce a vector RNA with terminal Rregions.

[0044] This producer DNA produces an RNA molecule that is packaged bythe lentiviral packaging system. The resulting vector particles willdeliver that RNA to a susceptible cell, the RNA will be converted to DNAby the lentiviral reverse transcriptase and it will be integrated intothe cells genome by the lentiviral integrase. The resulting proviruswill have the CMV promoter component of the producer DNA replaced by theshort lentiviral sequence from the end of the lentiviral U3 region andthe heterologous promoter that may confer tissue specific or regulatedgene expression. Because the lentiviral R region has been entirelyreplaced, there are no inhibitory TAR sequences in the integrated vectorgenome.

[0045] As will be evident, in order to function as a vector, thelentiviral LTR-deleted vector according to the invention will need tohave a reverse transcription system (compatible reverse transcriptaseand primer binding sites) and an integration system (compatibleintegrase and integration sites) allowing conversion to the provirus andintegration of the double-stranded DNA into the host cell genome.Usually these will include gag and pol proteins derived from theretrovirus. Additionally, the vector genome will need to contain apackaging signal. These systems and signals are described in more detailbelow in the Examples and will generally be provided by the retrovirus,on which the vector is based. That the vector particle according to theinvention is “based on” a retrovirus means that it is derived from thatretrovirus. The genome of the vector particle comprises components fromthat retrovirus as a backbone.

[0046] It will be evident also that, although the vector according tothe invention is based on a particular retrovirus, this may be agenetically or otherwise (e.g. by specific choice of packaging cellsystem) altered version of the retrovirus. For example, portions of theretroviral genome not required for its ability to be packaged, undergoreverse transcription and integrate, can be excluded. Also, the vectorsystem can be altered e.g. by using different env genes to alter thevector host range and cell types infected or transduced.

[0047] It may be advantageous to include further elements of theretrovirus on which the vector is based. For HIV this might includefunctional rev and RRE sequences, enabling efficient export ofRRE-containing RNA transcripts of the vector genome from the nucleus tothe cytoplasm of the target cell.

[0048] The selected NOI under the control of the exogenous promoter isor are chosen according to the effect sought to be achieved. For genetherapy purposes there will be at least one therapeutic NOI encoding aPOI which is active against the condition it is desired to treat orprevent. Alternatively or additionally, there may be a selected NOIwhich acts as a marker by encoding a detectable product. A NOI mayencode, for example, an anti-sense RNA, a ribozyme, a transdominantnegative mutant of a target protein, a toxin, a conditional toxin, anantigen that induces antibodies or helper T-cells or cytotoxic T-cells,a single chain antibody or a tumour suppresser protein.

[0049] Preferably, the retroviral vector according to the invention is asingle transcription unit vector, that is, the vector genome in DNA orRNA form is under the transcriptional control of no more than one vectorpromoter at any one time. In a preferred embodiment, this is achieved bylocating the polynucleotide sequence according to the invention suchthat in the DNA form of the vector genome integrated into the targetcell genome (the DNA provirus), it is under transcriptional control ofthe 5′ LTR. There are alternative ways of achieving a singletranscription unit vector, however. The vector genome could be designedas a self-inactivating vector (Yu et al., 1986 PNAS 83, 3194) in whichpart of the 3′ U3 sequences are deleted so that the transduced vectorgenome has a non-functional 5′ LTR promoter. The polynucleotide sequenceaccording to the invention would be operably linked to an internalconditional promoter between the LTRs which could be activated once thevector has transduced a target cell. Activation of the promoter might bedependent upon cellular or external factors.

[0050] Although single transcription unit vectors are preferred, othervectors are not excluded. It may be useful for example to include amarker gene in the vector, operably linked to a different promoter whichmay be active simultaneously with the promoter responsible fortranscription of the polynucleotide sequence encoding the fusionprotein. A marker gene encoding a selectable marker may be useful forselecting successfully transfected packaging cells, or successfullytransduced target cells. Marker genes encoding selectable markers may befor instance drug resistance genes or metabolic enzyme genes.

[0051] Where two or more NOI are present and under transcriptionalcontrol of the exogenous promoter, there may be an internal ribosomeentry site (IRES) e.g. from picornaviral RNA, to allow both NOI to beseparately translated from a single transcript. Retrovirusesincorporating IRES sequences have been constructed by others.

[0052] A further NOI may also be present under the control of a separatepromoter. Such a NOI may encode, for example, a selectable marker, or afurther therapeutic agent which may be among the therapeutic agentslisted herein. Expression of the NOI may be constitutive; in the case ofa selectable marker this may be useful for selecting successfullytransfected packaging cells, or for packaging cells which are producingparticularly high titers of the retroviral vector particles.Alternatively or additionally, the selectable marker may be useful forselecting cells which have been successfully infected with theretroviral vector and have the provirus integrated into their owngenome.

[0053] One way of performing gene therapy is to extract cells from apatient, infect the extracted cells with a retroviral vector andreintroduce the cells back into the patient. A selectable marker may beused to provide a means for enriching for infected or transduced cellsor positively selecting for only those cells which have been infected ortransduced, before reintroducing the cells into the patient. Thisprocedure may increase the chances of success of the therapy. Selectablemarkers may be, for instance, drug resistance genes, metabolic enzymegenes, or any other selectable markers known in the art.

[0054] However, it will be evident that for many gene therapyapplications of retroviral vectors, selection for expression of a markergene may not be possible or necessary. Indeed expression of a selectionmarker, while convenient for in vitro studies, could be deleterious invivo because of the inappropriate induction of cytotoxic T lymphocytes(CTLs) directed against the foreign marker protein. Also, it is possiblethat for in vivo applications, vectors without any internal promoterswill be preferable. The presence of internal promoters can affect, forexample, the transduction titres obtainable from a packaging cell lineand the stability of the integrated vector. Thus, single transcriptionunit vectors, which may be bi-cistronic or poly-cistronic, coding forone or two or more NOI, may be the preferred vector designed for use invivo.

[0055] It will be evident that the term “gene” is used loosely here, andincludes any nucleic acid of interest coding for a desired polypeptideof interest. Usually, the NOI delivered by the vector according to theinvention will be cDNAs.

[0056] The retroviral vector according to the invention may beconstructed according to methods known in the art. It is desirable thatthe retroviral vector genome does not encode any unnecessarypolypeptides, that is any polypeptides that are not required forachieving the effect the vector is designed for. In any case, theretroviral vector will be replication defective. Particular factors tobe taken into consideration when constructing a retroviral vectorinclude safety aspects and the avoidance of undesirable immuneresponses. Thus, it is necessary to exclude from the vector genome fulllength gag-pol or env coding regions, or preferably both. Preferably,the retroviral vector genome which will be inserted into the target cellin the form of a DNA provirus contains the minimum retroviral materialnecessary to function. This avoids both the possible reconstruction ofinfectious virus particles, and expression of unwanted virus proteins inthe target cell which could otherwise evoke undesirable immune responsesin the patient being treated.

[0057] The vector according to the invention will also be capable ofinfecting and transducing cells which are slowly-dividing, and whichnon-lentiviruses such as MLV would not be able to efficiently infect andtransduce. Slowly-dividing cells divide once in about every three tofour days. Mammalian non-dividing and slowly-dividing cells includebrain cells, stem cells, terminally differentiated macrophages, lungepithelial cells and various other cell types. Also included are certaintumour cells. Although tumours contain rapidly dividing cells, sometumour cells especially those in the centre of the tumour, divideinfrequently. The rate of cell division can easily be determined usingproliferation assays known in the art.

[0058] DNA constructs encoding the vector genome described herein arepreferably linked to a high efficiency promoter such as the CMVpromoter. Other high efficiency promoters are known. This gives rise toa high level of expression of the vector RNA in the host cell producingthe retroviral vector particles.

[0059] Suitable host or producer cells for use in the invention are wellknown in the art. Many retroviruses have already been split intoreplication defective genomes and packaging components. For those whichhave not the technology is available for doing so. The producer cellencodes the viral components not encoded by the vector genome such asthe gag, pol and env proteins. The gag, pol and env genes may beintroduced into the producer cell and stably integrated into the cellgenome to give a packaging cell line. The retroviral vector genome isthen introduced into the packaging cell line by transfection ortransduction to create a stable cell line that has all of the DNAsequences required to produce a retroviral vector particle. Anotherapproach is to introduce the different DNA sequences that are requiredto produce a retroviral vector particle e.g. the env coding sequence,the gag-pol coding sequence and the defective retroviral genome into thecell simultaneously by transient triple transfection (Landau & Littman1992 J. Virol. 66, 5110; Soneoka et al. 1995).

[0060] The strategy according to the invention has several advantages inaddition to those already described. Firstly, by making use of anon-lentiviral expression signal for a transcription unit it is possibleto make this vector genome a single transcription unit genome for bothproduction and expression in the transduced cell. This avoids the needfor internal promoters. The unpredictable outcome of placing additionalpromoters within the retroviral LTR transcription unit is welldocumented (Bowtell et al., 1988 J.Virol. 62, 2464; Correll et al., 1994Blood 84, 1812; Emerman and Temin 1984 Cell 39, 459; Ghattas et al.,1991 Mol.Cell.Biol. 11, 5848; Hantzopoulos et al., 1989 PNAS 86, 3519;Hatzoglou et al., 1991 J.Biol.Chem 266, 8416; Hatzoglou et al., 1988J.Biol.Chem 263, 17798; Li et al., 1992 Hum.Gen.Ther. 3, 381; McLachlinet al., 1993 Virol. 195, 1; Overell et al., 1988 Mol.Cell Biol. 8,1803;Scharfman et al., 1991 PNAS 88, 4626; Vile et al., 1994 Gene Ther 1,307; Xu et al., 1989 Virol. 171, 331; Yee et al., 1987 PNAS 84, 5197).The factors involved appear to include the relative position andorientation of the two promoters, the nature of the promoters and theexpressed nucleic acids and any selection procedures that may beadopted. The presence of internal promoters can affect both thetransduction titers attainable from a packaging cell line and thestability of the integrated vector. Loss of gene expression followingtransduction can be caused both by provirus deletions and reversibleepigenetic mechanisms of promoter shutdown. In addition, data fromtissue culture studies can often have no bearing on the performance ofthe vectors in vivo. These considerations suggest that simple retroviralvectors containing a single LTR promoter are likely to be promisingvectors for gene therapy (Correll et al., 1994 Blood 84, 1812). Inaddition, with the development of bi-cistronic vectors using only onepromoter (Adam et al., 1991 J.Virol 65,4985) it will also be possible toproduce single transcription unit vectors coding for two or more NOI,with correspondingly greater efficacy.

[0061] The second advantage of removing the HIV expression signalswithin the U3 and R regions is that these signals are subject to anumber of external influences on their activity. It is known that theHIV promoter can be activated by a variety of agents such as UV, stress,other viruses etc. (Peterlin 1992 in Human Retroviruses ed. Cullen. IRLPress) which makes the transcriptional status of the vector genomedifficult to control. Removal of these sequences will ensure greatercontrol over the nucleotide to be expressed.

[0062] In one embodiment, one or more NOI of interest is or are chosenaccording to the effect sought to be achieved. The fusion protein has oris capable of having the desired activity of the therapeutic geneproducts. The product encoded by one or more of the NOI may be anenzyme. The fusion protein may thus display the activity of one or moreenzymes. Where the NOI encode two different enzymes, the resultingfusion protein is a bifunctional enzyme. In the specific exampledescribed herein, the fusion protein comprises the enzymes tyrosinehydroxylase and DOPA dehydroxylase having enzyme activities as describedabove.

[0063] Preferably the NOI are linked by a sequence encoding a flexiblelinker. A suitable linker may comprise amino acid repeats such asglycine-serine repeats. The purpose of the linker is to allow thecorrect formation and/or functioning of the POI. It must be sufficientlyflexible and sufficiently long to achieve that purpose. Where the NOIencode two different enzymes, the linker needs to be chosen to allow thefunctioning of both of the enzymes. The coding sequence of the flexiblelinker may be chosen such that it encourages translational pausing andtherefore independent folding of the protein products of the NOI.

[0064] A person skilled in the art will be able to design suitablelinkers in accordance with the invention. Some specific examples ofsuitable linkers are given below; it will be evident that the inventionis not limited to these particular linkers.

[0065] 1. (Gly-Gly-Gly-Gly-Ser)3 as described in Somia et al., 1993 PNAS90, 7889.

[0066] 2. (Gly-Gly-Gly-Gly-Ser)5, a novel linker.

[0067] 3.(Asn-Phe-Ile-Arg-Gly-Arg-Glu-Asp-Leu-Leu-Glu-Lys-Ile-Ile-Arg-Gln-Lys-Gly-Ser-Ser-Asn)from HSF-1 of yeast, see Wiederrecht et al., 1988 Cell 54, 841.

[0068] 4.(Asn-Leu-Ser-Ser-Asp-Ser-Ser-Leu-Ser-Ser-Pro-Ser-Ala-Leu-Asn-Ser-Pro-Gly-Ile-Glu-Gly-Leu-Ser)from POU-specific OCT-1, see Dekker et al., 1993 Nature 362, 852 andSturm et al., 1988 Genes and Dev. 2,1582.

[0069] 5. (Gln-Gly-Ala-Thr-Phe-Ala-Leu-Arg-Gly-Asp-Asn-Pro-GlnGly) fromRGD-containing Laminin peptide, see Aumailly et al., 1990 FEES Lett.262,82.

[0070] 6.(Ser-Gly-Gly-Gly-Glu-Ile-Leu-Asp-Val-Pro-Ser-Thr-Gly-Gly-Ser-Ser-Pro-Gly)from LDV-containing linker, see Wickham et al., Gene Therapy 1995 2,750.

[0071] In addition to gene therapy, the invention has several otheruseful applications. The alteration of gene expression, by upregulatingor downregulating the production of gene products can be accomplishedusing the vectors of the invention. The vectors of the invention canalso be employed in vitro to produce therapeutic proteins, to expressselectable markers, or for other expression assays. Examples of proteinsthat may be expressed using the vectors of the invention include, butare not limited to, Factor VIII, Factor IX, erythropoietin, alpha-1antitrypsin, calcitonin, glucocerebrosidase, growth hormone, low densitylipoprotein (LDL) receptor, apolipoproteins (e.g. apolipoprotein E orapolipoprotein A-I), interleukins, interleukin receptors andantagonists, insulin, globin, immunoglobulins, catalytic antibodies,superoxide dismutase, immune responder modifiers, parathyroid hormone,interferons, growth factors, including insulin-like growth factors andnerve growth factors, tissue plasminogen activators, colony stimulatingfactors, and variants of these proteins.

[0072] In the particular embodiment described herein, the inventionaddresses the problems of the prior art by providing a single fusiongene that expresses a fusion protein composed of TH and DD. The singlegene encodes a single protein with both enzyme activities. This permitsthe construction of a simple single transcription unit retroviral vectorthat expresses both enzyme activities efficiently. The fusion gene isdesigned such that the enzymes are linked via a flexible linker, thecoding sequence of which has a short cluster of infrequently used codons(Haas et al., 1996 Curr. Biol. 6, 315) to encourage translationalpausing and, therefore, independent folding of the two domains of thenew bifunctional enzyme. Two different types of fusion gene were made.In the first the order of the enzyme activities is TH-DD and in theother it is DD-TH. Both types are made because they may have differentadvantages and properties under different conditions. Human tyrosinehydroxylase is encoded by a single gene which is alternatively splicedto create four types of TH that differ towards their amino terminus(Grima et al., 1987 Nature 326, 707; Kaneda et al., 1987 BBRC 146, 971).However, identical primers can be used to isolate all four cDNAs by PCRas the termini are the same.

[0073] The following examples are provided as a further description ofthe invention, and to illustrate but not limit the invention.

EXAMPLES Example 1

[0074] An HIV-Based LLD Vector with the MLV U3 Promoter and MLV RRegions.

[0075] Lentiviral vectors are particularly useful for gene transfer tonon-dividing cells. Amongst many important non-dividing target cells arethe neurons of the human brain. These cells might be target cells forthe delivery of thdd or ddth cells for the treatment of Parkinson'sdisease. This Example describes the construction of an HIV based vectorwhich will deliver and express thdd or ddth genes, for example.

[0076] The structure of a general HIV LLD vector system is shown in FIG.2. This example is shown in FIGS. 3 and 4. It is constructed as follows.the minimal requirements for HIV reverse transcription are the primerbinding site (PBS) to initiate the negative strand DNA synthesis, thepolypurine tract (PPT) to initiate the positive DNA synthesis, andidentical 5′ and 3′ R sequences to allow the first template switch. Theincorporation of the PBS and PPT from HIV-1 into the vector and the Rsequences from MLV into both LTRs is therefore required. As secondarystructure within the 5′ U5 region might be important for reversetranscription, the U5 region in the 5′ LTR is from HIV-1. For the U5region at the 3′ LTR, the U5 from HIV-1 was used to make sure correcttermination of transcription occurred at the R-U5 border. However, anytermination signals could be used. For efficient integration, 30nucleotides at the 5′ end of the HIV-1 U3 at the 3′ LTR wereincorporated.

[0077] In order for the MLV U3 element to appear in the 5′ LTR afterreverse transcription, it must be in the 3′ LTR of the viral RNA. Thewhole MLV U3 except 30 bps of the 5′ end replaced the HIV-1 U3. The 3′LTR of the vector was designed to contain several convenient restrictionsites, so that the MLV U3 can be easily replaced by other heterologouspromoters (FIG. 4). Any heterologous promoters will be amplified by PCRwith primers containing StuI and NarI sites at each end and will be usedto replace the MLV U3. Not only StuI but also NheI and Afill may be usedat the 5′ end of the promoter cassettes. NarI(GGCGCC) is located on thejunction between the promoter and R, so that the transcription startsite from the heterologous promoter can be preserved. The MLV U3sequences between Xbal and NarI contains the basic promoter elementsincluding TATA box, GC box, and CAAT box. Therefore the MLV enhancer canbe replaced by any other enhancers as a StuI (or NheI or AfIII)-XbaIcassettes.

[0078] For efficient packaging 353 nucleotides of gag is known to besufficient (Srinivasakumar et al., 1996 CSH Retrovirus Meetingabstract). The 353 nucleotides of gag sequences corresponds to thesequences from 790 to 1144, within this three ATG's (790, 834, 894) wereremoved by mutation. In addition a polycloning site is locateddownstream of gag.

[0079] In order to achieve efficient export of RNA species encoded byHIV genome, rev and RRE, are required. They are included in the LLDvector and correspond to sequences 5861 to 6403 and 7621 to 9085 from isHIV-1 (HXB2). Tat coding sequence is not present in the vector.

Details of Construction of the Producer DNA

[0080] A. 5′ Structure (All HIV-1 Coordinates are from HXB from the LosAlamos Sequence Database and MoMLV Sequences are from Shinnick et al.1981 Nature 293. 543)

[0081] The 5′ half of the vector contains the hybrid 5′ LTR (CMVpromoter-MLV R-HIV-1 U5), HIV-1 PBS, and HIV-1 packaging signal. Thiswill be constructed by recombination PCR. One of the templates for thePCR, pHIVdge2, is an HIV-1 proviral DNA which has a mutation created byfilling-in and religation at the ClaI site (831) and a deletion betweenNdeI(6403) and BgIII(7621). The junction between MLV R and HIV-1 U5 iscreated by two primary PCR reactions (using the primer NIT1 and NIT2;NIT3 and NIT4) and a secondary PCR reaction (using the primers NIT1 andNIT4). The PCR product is inserted into pBluesriptKS+ (STRATAGEN) atKpnI and XhoI site (Construct A1). In order to mutate three ATGs in thegag region, the primers contain mutated codons. NIT1:5′-ccgggtacccgtattcccaataaagcctcttgctgtttgca-3′ (SEQ ID NO:1) NIT2:5′-ctacgatctaattctcccccgcttaatactgacgctctcgcacctatctc-3′ (SEQ ID NO:2)NIT3:5′-gcgggggagaattagatcgtagggaaaaaattcggttaaggccagggggaaagaaaaaatataaatt(SEQ ID NO:3) aaaacatatagtttggg-3′ NIT4:5′-gaattctcgaggcgtgctgtgcttttttctatc-3′ (SEQ ID NO:4)

[0082] The CMV promoter-MLV R fragment is amplified by PCR from pRV109(Soneoka et al., 1995 Nucl. Acids Res. 23, 628) to contain KpnI sites atboth ends using the PCR primers NIT5 and NIT6 and inserted intoconstruct A1 to produce construct A2. (SEQ ID NO:5) NITS:5′-gtaggtacccgttacataacttacggtaaatg-3′ (SEQ ID NO:6) NIT6:5′-agaggctttattgggaatacg-3′

[0083] B. 3′ Structure

[0084] The 3′ half of the vector genome includes the HIV-1 rev codingregion and RRE, PPT, 36 by of 5′ end of HIV-1 U3, and the whole MLV LTRexcept 30 by of 5′end. The sequences (5861-6000) are PCR amplified frompHIVdge2 (using NIT7 and NIT8) and are subcloned into pSP64 (PROMEGA) atBamHI and SacI site (Construct B1). (SEQ ID NO:7) NIT7:5′-cacggatccactagttggaagcatccaggaagtcagc-3′ (SEQ ID NO:8) NIT8:5′-ctctgactgttctgatgagc-3′

[0085] The SacI-SacI fragment (6000-6403 and 7621-9572) from pHIVdge2 isinserted into the above construct to produce construct B2. Finally theHIV-1-MLV hybrid LTR will be created by two primary PCRs (using NIT9 andNIT10 with pHIVdge2 as the template; NIT11 and NIT12 with pLXSN(Accession number M28248; Miller et al., 1989) as the template) and onesecondary PCR reaction (using NIT9 and NIT12). The PCR product will beinserted at the XhoI and EcoRI sites in Construct B2 to produceConstruct B3. NIT9: 5′-gagcagcatctcgagacctgg-3′ (SEQ ID NO:9) NIT10:5′-tggcgttacttaagctagcaggcctgtcttctttgggagtgttttagc-3′ (SEQ ID NO:10)NIT11: 5′-cccaaagaagacaggcctgctagcttaagtaacgccatttttcc-3′ (SEQ ID NO:11)NIT12: 5′-cctgaattccgcggaatgaaagacccccgctgacg-3′ (SEQ ID NO:12)

[0086] C. Complete Vector

[0087] The two halves of the vector are combined by inserting theSpeI-SacII fragment from construct B3 into construct A2. The resultingconstruct, C1, possesses a poly-cloning site;XhoI-SaII-ClaI-HindIII-EcoRV-EcoRI-PstI-SmaI-BamHI-SpeI(underlined sitesare unique in the vector). This plasmid is designated pLLD1 and theretroviral vector that it produces is LLD1.

[0088] The β-galactosidase gene was then taken from pSP72-lacZ(XhoI-BamHI) and inserted into the construct C1 at SaII and BamHI toproduce LLD1-lacZ. This was used to transfect 293T cells together withplasmids providing the HIV gag and pol components (pRV664, FIG. 5) andeither a plasmid expressing gp160 from HIV (pRV438 or pSynp160 mn, FIG.5) or a plasmid expressing the VSVG protein (pRV67, FIG. 5). Anyplasmids encoding the same proteins would work equally well. Theresulting virus that is produced transduced the lacZ gene to CD4+ Helacells in the case of virus containing gp160 and to CD4− Hela cells inthe case of the VSVG bearing virus. In addition the VSVG bearing virusdelivers lacZ to post-mitotic neurones. In each case the expression ofthe lacZ gene is high, as determined by Xgal staining, and independentof Tat.

[0089] Alternatively, ddth1 is used to illustrate the principle of afusion construct, but any of the fusion genes could be used. Plasmid pX1is cut with HincII and SpeI and the fragment purified. This is theninserted into LLD1 cut with EcoRV and SpeI to create pLLD1:thdd1. Whenthis is transfected into a packaging cell line (suitable packagingcomponents are shown in FIG. 5) and viral vector particles produced,those vector particles deliver the thdd gene to the recipient cellswhere the fusion enzymes are expressed. Such a retroviral vector systemis useful for the treatment of Parkinson's disease by gene therapy.

Example 2

[0090] Other LLD Vectors.

[0091] Systems similar to that described in Example 1 can be producedfrom other lentiviruses. These systems avoid using HIV, with itsassociated perceived risks as a gene delivery system. For exampleconstructions could be designed using sequence information from FIV(Talbott et al., 1989 PNAS 86, 5743), EIAV (Payne et al., 1994J.Gen.Virol. 75, 425), Visna virus (Sonigo et al., 1985 Cell 42, 369;Querat et al., 1990 Virology 175, 434), BIV (Garvey et al., 1990Virology 175, 391), CAEV (Saltarelli et al., 1990 Virology 179, 347) andSIV (Los Alamos sequence database).

Example 3

[0092] Construction of TH-DD Fusion Genes Designated thdd1-4 (FIG. 7).

[0093] A human brain Substantia nigra cDNA library (Clontech. HI-3009a &b) is used as template DNA in a PCR amplification of the TH and DDcDNAs. The primers are shown in FIG. 9. In the case of the four TH cDNAsrepresenting the HTH-1 to HTH-4 genes (Grima et al.; Kaneda et al.),they are all treated in the same way from a pool of PCR products andthen identified after cloning and sequencing. The TH PCR products areproduced from linkers containing a HincII site at the 5′ end of thenucleic acid and a flexible linker and HindIII site at the 3′ end. Theflexible linker amino acid sequence is (Gly₄-Ser)₃, a sequence oftenused to link the two chains of an antibody to produce an scFv (e.g.Somia et al., 1995 PNAS 92, 7570). The human DD PCR product was designedto have a HindIII site at the 5′ end and a SpeI site at the 3′ end. Thetwo fragment are ligated and the ligated products of the correct size(2.98 kb, 2.99 kb, 3.06 kb and 3.07 kb for the four variants) arepurified from an agarose gel. The purified fragments are then insertedinto pBLUEscriptKS+ using HincII and SpeI. This ligation mixture is usedto transform E.coli (XL2-Blue ex. Stratagene 200249) and clones wereused to prepare DNA which is then sequenced to ensure that the genes areintact and to identify HTH1-4. Plasmids containing fragments encodingthe four different HTH coding sequences fused to DD are designatedpthdd1-4. The the HincII-SpeI fragments from these plasmids are theninserted into the mammalian expression vector pC1-neo (Promega E1841).This is achieved by cutting pC1-neo with XhoI and SmaI and cutting thepBluescriptKS+ derived plasmids with SpeI and blunt ending and thencutting with XhoI. The cut products are then ligated together andcorrect plasmids checked by minipreps. The pC1-neo plasmids containingthe fusion genes are designated pC1thdd1-4. These are then used totransiently transfect 293T cells which are then assayed for TH and DD bythe methods of Waymire et al. (1971) (AnaI.Biochem. 43, 588) and themethod described in Anal. Biochem. (1984139, 73). In each casesignificantly increased levels of TH and DD are seen compared withcontrol cells transfected only with pC1-neo. This demonstrates that thefusion genes expresses fusion proteins with both activities.

Example 4

[0094] Construction of DD-TH Fusion Genes Designated ddth1-4 (FIG. 8).

[0095] The construction of these genes is identical to that of Example 3but the DD and TH coding sequences are in reciprocal locations.Similarly dual enzyme activities are encoded by the ddth1-4 genes.

Example 5

[0096] Construction of a Retroviral Vector Expressing a thdd Gene is aSingle Transcription Unit Configuration.

[0097] The thdd and ddth genes are useful for the gene therapy ofParkinson's disease. They can be used in a wide range of vectors butthey are particularly suited to single transcription unit retroviralvectors. An example of such a vector is produced as follows: Startingwith pLNSX (Miller) a polylinker is inserted into the vector. Briefly, aSspI/HindIII fragment, containing the polylinker from pBluescriptKS+ isinserted into pLNSX cut with SspI and HindIII. The resulting plasmid isknown as pMLD1. Plasmid, pX1, for example, is then cut with SpeI andthen the ends filled in with DNA polymerase. The plasmid is then cutagain with XhoI. The resulting thdd fragment is then inserted into pMLD1cut with XhoI and ClaI (blunt-ended) to produce the resulting moleculepMLD1:thdd1. When this plasmid is used to transfect a packaging cellline retroviral vectors are produced which transduce susceptible cellswith the thdd gene in a single transcription unit configuration. In thiscase the gene is expressed from the MLV LTR promoter but any promoterinserted into the 5′ LTR via a U3 replacement or similar strategy wouldbe equally effective.

[0098] Having thus described in detail preferred embodiments of thepresent invention, it is to be understood that the invention defined bythe appended claims is not to be limited to particular details set forthin the above description, as many apparent variations thereof arepossible without departing from the spirit or scope of the presentinvention. Modifications and variations of the method and apparatusesdescribed herein will be obvious to those skilled in the art, and areintended to be encompassed by the following claims.

1 20 1 41 DNA Unknown Primer NIT1 used in PCR reaction to createjunction between MLV R and HIV-1 U5 1 ccgggtaccc gtattcccaa taaagcctcttgctgtttgc a 41 2 50 DNA Unknown Primer NIT2 used in PCR reaction tocreate junction between MLV R and HIV-1 U5 2 ctacgatcta attctcccccgcttaatact gacgctctcg cacctatctc 50 3 84 DNA Unknown Primer NIT3 used inPCR reaction to create junction between MLV R and HIV-1 U5 3 gcgggggagaattagatcgt agggaaaaaa ttcggttaag gccaggggga aagaaaaaat 60 ataaattaaaacatatagtt tggg 84 4 33 DNA Unknown Primer NIT4 used in PCR reaction tocreate junction between MLV R and HIV-1 U5 4 gaattctcga ggcgtgctgtgcttttttct atc 33 5 32 DNA Unknown PCR primer NIT5 used in amplificationof CMV promoter 5 gtaggtaccc gttacataac ttacggtaaa tg 32 6 21 DNAUnknown PCR primer NIT6 used in amplification of CMV promoter 6agaggcttta ttgggaatac g 21 7 37 DNA Unknown PCR primer NIT7 used inamplification of pHIVdge2 7 cacggatcca ctagttggaa gcatccagga agtcagc 378 20 DNA Unknown PCR primer NIT8 used in amplification of pHIVdge2 8ctctgactgt tctgatgagc 20 9 21 DNA Unknown PCR primer NIT9 used toprepare HIV-1-MLV hybrid LTR 9 gagcagcatc tcgagacctg g 21 10 48 DNAUnknown PCR primer NIT10 used to prepare HIV-1-MLV hybrid LTR 10tggcgttact taagctagca ggcctgtctt ctttgggagt gttttagc 48 11 44 DNAUnknown PCR primer NIT11 used to prepare HIV-1-MLV hybrid LTR 11cccaaagaag acaggcctgc tagcttaagt aacgccattt ttcc 44 12 35 DNA UnknownPCR primer NIT12 used to prepare HIV-1-MLV hybrid LTR 12 cctgaattccgcggaatgaa agacccccgc tgacg 35 13 33 DNA Unknown TH5-1 mammalian primerused to amplify the tyrosine hydroxyase (TH) gene from cDNA library 13cacagtcgac catgcccacc cccgacgcca cca 33 14 77 DNA Unknown TH3-1mammalian primer used to amplify the tyrosine hydroxyase (TH) gene fromcDNA library 14 cgtacaagct tcgatcctcc acctcccgag ccacctccgc ctgaaccgcctccaccgcca 60 atggcactca gcgcatg 77 15 32 DNA Unknown DD5-1 mammalianprimer used to amplify the DOPA decarboxylase (DD) gene from cDNAlibrary 15 acgcaaagct tatgaacgca agtgaattcc ga 32 16 34 DNA UnknownDD3-1 mammalian primer used to amplify the DOPA decarboxylase (DD) genefrom cDNA library 16 ctggactagt ctactccctc tctgctcgca gcac 34 17 32 DNAUnknown DD5-2 mammalian primer used to amplify the DOPA decarboxylase(DD) gene from cDNA library 17 cacagtcgac catgaacgca agtgaattcc ga 32 1877 DNA Unknown DD3-2 mammalian primer used to amplify the DOPAdecarboxylase(DD) gene from cDNA library 18 cgtacaagct tcgatcctccacctcccgag ccacctccgc ctgaaccgcc tccaccctcc 60 ctctctgctc gcagcac 77 1933 DNA Unknown TH5-2 mammalian primer used to amplify the tyrosinehydroxyase (TH) gene from cDNA library 19 acgcaaagct tatgcccacccccgacgcca cca 33 20 34 DNA Unknown TH3-2 mammalian primer used toamplify the tyrosine hydroxyas (TH) gene from cDNA library 20 ctggactagtctagccaatg gcactcagcg catg 34

We claim:
 1. A lentiviral vector capable of transducing a non-dividingor slowly-dividing cell, said vector comprising a lentiviral LTR-deletedvector.
 2. The vector according to claim 1, further comprising anucleotide sequence encoding a protein of interest.
 3. A method forproducing a protein of interest in a non-dividing or slowly-dividingcell, comprising the steps of: a) transducing the cell with the vectoraccording to claim 2; and b) expressing the protein of interest in thecell.
 4. The method according to claim 3, wherein the non-dividing cellis a neuron.
 5. A target cell in vitro comprising the vector of claim 1.6. A target cell in vitro comprising the vector of claim
 2. 7. A methodof performing gene delivery on a target cell comprising the steps of: a)transducing the target cell with the vector according to claim 2; and b)delivering the nucleotide sequence to the target cell.